NOXA Is Important for Verticillium dahliae’s Penetration Ability and Virulence

NADPH oxidase (Nox) genes are responsible for Reactive Oxygen Species (ROS) production in living organisms such as plants, animals, and fungi, where ROS exert different functions. ROS are critical for sexual development and cellular differentiation in fungi. In previous publications, two genes encoding thioredoxin and NADH-ubiquinone oxidoreductase involved in maintaining ROS balance were shown to be remarkably induced in a highly versus a weakly aggressive Verticillium dahliae isolate. This suggested a role of these genes in the virulence of this pathogen. NoxA (NADPH oxidase A) was identified in the V. dahliae genome. We compared in vitro expression of NoxA in highly and weakly aggressive isolates of V. dahliae after elicitation with extracts from different potato tissues. NoxA expression was induced more in the weakly than highly aggressive isolate in response to leaf and stem extracts. After inoculation of potato detached leaves with these two V. dahliae isolates, NoxA was drastically up-regulated in the highly versus the weakly aggressive isolate. We generated single gene disruption mutants for NoxA genes. noxa mutants had significantly reduced virulence, indicating important roles in V. dahliae pathogenesis on the potato. This is consistent with a significant reduction of cellophane penetration ability of the mutants compared to the wild type. However, the cell wall integrity was not impaired in the noxa mutants when compared with the wild type. The resistance of noxa mutants to oxidative stress were also similar to the wild type. Complementation of noxa mutants with a full length NoxA clones restored penetration and pathogenic ability of the fungus. Our data showed that NoxA is essential for both penetration peg formation and virulence in V. dahliae.

Verticillium klebahnii and V. isaacii Isolates Exhibit Host-dependent Biological Control of Verticillium Wilt Caused by V. dahliae

Verticillium dahliae, the soil-borne fungal pathogen, causes vascular wilt on many economically important crops resulting in significant yield losses. Verticillium klebahnii (isolate PD659) and V. isaacii (isolate PD660), two related species that cause little or no symptoms in some hosts, were evaluated as potential biocontrol agents (BCA) in eggplant, lettuce, and tomato by pre, post, and co-inoculation with a virulent race 1 isolate of V. dahliae (VdLs16). Initial studies demonstrated that the biocontrol efficacy of both BCAs was similar to reference BCA Talaromyces flavus (NRRL15936) across all hosts (α= 0.05). Subsequent experiments with PD659 against V. dahliae isolate Sm113 from eggplant, VdLs16 and VdLs17 isolates from lettuce, and Le1811 isolate from tomato demonstrated a significant biocontrol efficacy in eggplant and tomato but not in lettuce (at 95% CI), suggesting host-dependent effectiveness of V. klebahnii. Confocal microscopy using GFP-tagged tomato V. dahliae isolate Le1811 indicated delayed xylem colonization or lack of pathogen progression into the vascular system in a host-dependent manner on BCAs treated plants. Quantitative analyses of the expression of defense-related genes PR1a, PR5, acidic extracellular b-1,3-glucanase (GlucA), basic intracellular b-1,3-glucanase (GlucB), acidic extracellular chitinase (Chi3), basic intracellular chitinase (Chi9), and cysteine proteases (cysProreases) in tomato in the presence or absence of PD659 suggested an elevated expression of defense-related genes in compatible interaction of V. dahliae-tomato cv. Early Pak. Verticillium klebahnii (PD659) may delay the entry of V. dahliae by competing for space or nutrients during the initial stages of root colonization.

Susceptibility of Weedy Hosts from Pacific Northwest Potato Production Systems to Crop-Aggressive Isolates of Verticillium dahliae

Verticillium wilt, caused by Verticillium dahliae, is a disease of dicotyledonous crops such as potato and has a wide host range and persistent, long-term survival structures called microsclerotia that can persist in soil for up to 14 years. Some V. dahliae isolates are particularly aggressive on a specific plant host while retaining the ability to infect a wide range of other hosts. Weeds can serve as hosts for V. dahliae but whether they serve as sources of inoculum for aggressive isolates of V. dahliae to crop hosts is unknown. The goal of this research was to quantify V. dahliae microsclerotia obtained from 16 weeds which were grown in the greenhouse. Potting medium was infested with one of eight V. dahliae isolates from potato, mint, sugar beet, sunflower, tomato, and watermelon. The isolates from mint and potato were aggressive on the host from which they were originally isolated. All 16 weeds were infected by at least one V. dahliae isolate, although the number of microsclerotia produced from some infections was relatively low (≤5 microsclerotia/g of dry plant). Black nightshade yielded greater numbers of microsclerotia of the V. dahliae potato isolate than any other isolate in three of four trials in the greenhouse (second trial false discovery rate, adjusted P ≤ 0.0158; third trial, P ≤ 0.0264; and fourth trial, P ≤ 0.0193). Litchi tomato yielded greater numbers of microsclerotia of the V. dahliae potato isolate than any other isolate in one of four trials (first trial, P ≤ 0.0149). A V. dahliae isolate from tomato yielded greater numbers of microsclerotia in large crabgrass and wild oat in a second trial (P ≤ 0.0158). Weeds, depending on the species, grown during and between potato crop rotations may increase the number of microsclerotia of the potato-aggressive isolates of V. dahliae.

Symptomless Host and Nonhost Responses of Paulownia (Paulownia spp.) to Olive-Defoliating Verticillium dahliae

Symptomless host and nonhost responses of Paulownia spp. to olive-defoliating (D) Verticillium dahliae is reported for the first time. Two paulownia clones, Paulownia elongata ‘PC-2’ and P. elongata × P. fortunei ‘PC-3’, were inoculated with a V. dahliae isolate representative of the D pathotype by either root dip or stem injection with a conidial suspension, repeated transplanting to a V. dahliae-infested soil mixture, or root dip in the conidial suspension followed by transplanting to the infested soil mixture. ‘Picual’ olive and ‘Sugar Baby’ watermelon were included in all experiments as susceptible standards to show that the inoculation procedures and incubation conditions were successful. Plants were incubated under conditions optimal for Verticillium wilt that caused severe disease in ‘Picual’ olive and ‘Sugar Baby’ watermelon in the growth chamber, shade house, and field microplots for 30 to 57 weeks in three independent experiments. No foliar symptoms developed on paulownia, whose stems were found free of V. dahliae both by isolation on semiselective NP-10 medium as well as by a nested-polymerase chain reaction assay using total genomic DNA from inoculated plants that effectively detected D V. dahliae in olive stems. V. dahliae was isolated to a limited extent from roots of PC-3 paulownia plants after 30 weeks of growth in the infested soil mixture but not from those that were root-dip inoculated or from PC-2 plants regardless the method of inoculation. The symptomless host and nonhost responses of Paulownia spp. to D V. dahliae may have practical applications in the use of fertile soils in southern Spain, particularly in those that are highly infested with the highly virulent D pathotype, as well as a replacement crop for Verticillium wilt-affected olive orchards in that region.

First Report of Verticillium Wilt of Pistachio Caused by Verticillium dahliae in Spain

Pistachio (Pistacia vera L.) trees in the Castilla La Mancha and Andalusia regions of central and southern Spain are grown close to olive orchards, which are often severely affected by Verticillium dahliae. During the last decade, wilt and death of one or several branches have been observed on pistachio (cv. Kerman) scions grafted on rootstock (P. terebinthus). Discoloration of vascular tissue was occasionally observed. In five surveyed orchards, incidence of affected trees was less than 2%. Wood chips with the bark removed from symptomatic trees were washed in running tap water, surface disinfested in 0.5% sodium hypochlorite for 1 min, and placed onto potato dextrose agar plates incubated at 25°C in the dark. Isolates were identified as V. dahliae on the basis of the characteristics of microsclerotia, conidiophores, and conidia. V. dahliae isolate V117 from olive was used as reference (1). The fungus was also isolated from soil in pistachio orchards using wet sieving and a modified sodium polypectate agar medium (1). Inoculum density varied from 0 to 4.73 microsclerotia per gram of soil. P. terebinthus seedlings were inoculated with two isolates of V. dahliae from pistachio trees by injecting the stems with 50 μl of a conidial suspension (107 conidia per ml) (2). Wilt symptoms of varying severity developed in 12 and 15 seedlings of the 20 pistachio seedlings inoculated with each of two isolates. No symptoms developed on the control seedlings. The pathogen was recovered from stem tissues of inoculated plants. To our knowledge, this is the first report of Verticillium wilt of pistachio in Spain. This study demonstrates the susceptibility of certain rootstocks to V. dahliae and the importance of using resistant rootstocks, such as UCBI (2), in pistachio plantations established on soils infested by V. dahliae. References: (1) F. J. López-Escudero and M. A. Blanco-López. Plant Dis. 91:1372, 2007. (2) D. P. Morgan et al. Plant Dis. 76:310, 1992.

Efficacy of seaweed concentrate from Ecklonia maxima (Osbeck) and conventional fungicides in the control of Verticillium wilt of pepper

In order to control the causal agent of Verticillium wilt of pepper (Verticillium dahliae), the efficacy of two conventional fungicides, thiophanate-methyl and carbendazim, and seaweed concentrate (SWC) from Ecklonia maxima was evaluated in greenhouse conditions. Pepper plants were inoculated with selected V. dahliae isolate in the stage of more than nine fully developed leaves on primary stem. The tested fungicides and SWC were applied three days before inoculation of pepper plants. Carbendazim was the most efficient fungicide among tested substances (69.64%). SWC proved to be more effective when applied at 1.0% concentarion (41.96%). The use of thiophanate-methyl provided good Verticillium wilt control in pepper (60.71%). SWC was less efficient than thiophanate-methyl and carbendazim, but still significantly better compared to the disease control plot.

Verticillium Wilt of Paprika Caused by a Highly Virulent Isolate of Verticillium dahliae

Verticillium dahliae caused wilting, stunting and early dying of paprika plants in Israel, resulting in a 22% reduction in yield. A V. dahliae isolate from diseased paprika plants was slower growing on agar medium than V. dahliae isolates from potato, tomato, watermelon, and Dodonaea, but formed microsclerotia earlier. The paprika isolate of V. dahliae was highly virulent to paprika in pathogenicity tests. Disease severity indices obtained from three paprika cultivars ranged from 3.7 to 4.9 (on a scale of 0 to 5). The disease caused height reductions of 43 to 62% in the three inoculated paprika cultivars, and pathogen colonization levels were 10- to 35-fold higher in stems than in leaves. The potato isolate of V. dahliae did not produce symptoms or cause stunting of paprika, and could not be detected in stems or leaves. The tomato, watermelon, and Dodonaea isolates did not cause disease symptoms in paprika, although they colonized the plant tissue. In potato and eggplant, the paprika isolate caused a higher incidence of disease with more severe symptoms and a higher level of tissue colonization than did the potato isolate.